1. Field of the Invention
The present invention generally concerns blood clotting components and mechanisms.
The present invention particularly concerns new devices and methods for studying clotting mechanisms and factors. More specifically, an apparatus and method for measuring and monitoring health and activity of platelets and other clotting factors are described. Most specifically, a clot retractometer and its method of use are provided to measure clot contractility forces as a means to provide a single point “funnel detection” procedure useful in aiding physiological and clinical research and patient diagnosis and monitoring of many diseases, as well as screening populations.
2. Description of Related Art
Introduction
Everyone has seen a clot form as a result of injury to tissue, such as, for example, a scrape, puncture or a bleeding nose. However, the formation of a clot is a complex, cascading process that is still not completely elucidated, either physiologically or clinically. The clotting phenomenon, or lack thereof, is manifest in numerous clinical conditions, and is relevant to their prognosis.
In response to soft tissue injury, the haemostatic mechanism is activated to stop bleeding and restore vascular integrity. Blood protein and cellular interactions lead to the formation of a platelet plug and ultimately generation of clot comprising platelets and protein fibers. These reactions have to occur rapidly because the amount of blood lost is dependent on the time required to arrest the bleeding. Although rapid stoppage of blood loss is critical in some cases, inappropriate induction of clotting can have devastating effects such as decreased blood flow to the organs and resultant ischemic damage, such as heart attacks and stroke if the clot is not solubilised. To counterbalance these potentially severe consequences the haemostatic system is uses certain clotting inhibitors, and clot-dissolving enzymes.
Following vascular damage, the exposure of flowing platelets to the subendothelial lining allows the establishment of adhesive interactions with the immobilized surfaces. Platelets then become activated due to contact with thrombogenic substrates and stimulation by locally released or generated agonists. Subsequent platelet deposition relies on the binding of plasma-soluble adhesive molecules, and on the externalization of adhesive molecules from the platelets' granular reservoirs, this process conditions the newly recruited monolayer of platelets to become the reactive surface for continuing platelet accrual.
Immediately after platelet arrest, the clotting process begins by the participation of platelet released substances and fluid phase coagulation factors. Initiation of the coagulation cascade results in the conversion of prothrombin to thrombin (a serine protease). Thrombin cleaves two pairs of peptides (fibrinopeptides A and B) from the aminoterminal ends of the Aα and Bβ chains of the fibrinogen molecule. Cleavage of fibrinopeptide-A is sufficient to initiate clot assembly (4). The monomer units formed initiate a self-assembly process of forming protofibrils. Weak lateral interactions between protofibers increase as the protofibers lengthen, resulting in their alignment and coalescence, to ultimately yield fibers. This process leads to the formation of a network composed of fibrin polymers and spaces filled with fluid. Once the fibrin network is formed, the platelets begin to contract, resulting in a pull on the strands of the fibrin network. Platelet contraction requires active restructuring of the platelet cytoskeleton.
Dynamic rearrangements in the cytoskeleton are crucial during platelet activation in both, initial platelet adhesion to surfaces (see FIG. 1) and platelet to platelet cohesion. Actin polymerization in non-stimulated platelets is limited by monomer-sequestering proteins such as thymosin β4, profilin and barbed end-capping proteins such as gelsolin (5-7). Under these conditions, around 2,000 actin filaments are distributed in the cytoskeleton and in the membrane skeleton right under the inner surface of the plasma membrane (8). After the stimulation by strong agonists, there is a rapid increase in actin polymerization, with reorganization of the two actin networks, resulting in a change of shape with formation of filopodia and lamellipodia at the cell periphery. This is followed by redistribution of actin and other cytoskeletal and signaling proteins form the membrane skeleton to the cytoskeleton (9;10). Platelet spreading is associated with the appearance of actin stress fibers and focal-adhesion-like structures that contain clusters of integrins and vinculin (11). Small GTPases of the Rho family—such as cdc42Hs, Rac, and Rho—have been implicated in the formation of filopodia, lamellipodia and focal adhesion plaques in many cell types (12), and the same may occur in platelets.
Under normal conditions, the coagulation system remains in a fine balance. Pathologic alterations of the system may induce a risk of hemorrhage or increase the potential for thrombosis. An example of the former would be the bleeding disorder of hemophilia, which results from a low activity of Factor VIII, a blood clotting protein. An example of the latter would be recurrent venous thrombosis in individuals who have decreased levels of the coagulation inhibitor antithrombin III. Patients with decreased ability to remove clots, decreased fibrinolytic potential, are also at risk for thrombosis
To counterbalance the above mentioned mechanisms that precipitate platelet activation, platelets are downregulated by the anti-thrombotic potential of normal endothelial cells, in vivo, and by substances produced by the activated platelets. The majority of pathways that result in inhibition of platelet aggregation and procoagulant activities act by increasing the internal level of cyclic AMP, which activates the cyclic AMP-dependent protein-kinase. This leads to serine-threonine phosphorilation of an array of substrates.
Experimentally, the result of the platelet contraction and the tension applied on the fibrin network strands is observed in vitro as clot retraction. Macroscopically, clot retraction is seen as a dramatic reduction in clot volume in a process that expels the fluid trapped inside the clot. Although the physiological role of clot retraction is not completely understood, it is assumed that it helps in approximating the edges of a tissue defect and in concentrating the clot in the area of injury (4). One issue that it is clear in clot retraction is that in order for this process to occur normally all haemostatic mechanisms must act in synchrony. The two primary requirements for proper clot retraction to occur are the formation of an appropriate fibrin network and healthy platelets, capable of contracting and anchoring the fibrin network. The structure and formation of the fibrin network are sensitive to pH, ionic strength, calcium concentration, plasma proteins, platelet release products, leukocyte materials, etc. (4).
Examples of pathological conditions that affect the structure of the fibrin network are diabetes mellitus and multiple myeloma (4;13). Healthy platelets need to express the integrin αIIbβ3 on their surface to properly anchor the fibrin strands and they need to be metabolically fit for the task. Examples of pathological conditions that affect platelet metabolism are diabetes mellitus and uremia (14). Also, the age of platelets affects their performance, this aspect is particularly important for transfusion purposes.
The wide spectrum of processes involved in clot retraction, including biochemical, biorheological and biomechanical mechanisms, in conjunction with fine controlling and orchestration mechanisms, makes clot retraction a very desirable focus point that represents the well-being of all the steps required for this event to take place. This approach of “funnel detection” yields excellent means for population screening and individual patient monitoring for clinical progress.
Current State-of-the-Art
In clinical practice, the measurement of platelet viability has been used mainly to set standards for appropriate storage and handling of platelet concentrates. These techniques include estimation of the life-span after storage with radiolabeling, measuring the reduction of bleeding time, and semi-quantitative estimation of platelets to form aggregates in vitro with the use of platelet aggregometers. These techniques, however, are not routinely used to evaluate platelet performance. A more practical estimation of the capacity of platelets to function normally appears to be retention of shape, ATP content and function in the osmotic reversal reaction (15).
Methods Currently Used to Evaluate Clot Retraction
A common method utilized to evaluate clot retraction is quantitation of the fluid volume expelled by the clot during retraction, and estimation of the volume of the residual clot (16). This is a qualitative essay that does not provide information about the force generated during clot retraction.
Another known method involves the formation of cylinders or strips of clots, which are then immobilized on one end and anchored to a force transducer (17) on the other. This technique requires mechanical manipulation of the sample and bathing of the clots in a foreign substance that may alter the natural process of clot retraction.
Yet another technique utilizes a rheometer to measure the normal force development during clotting and retraction (18). An important limitation of this technique is the high cost of the equipment.
A method described by Carr in U.S. Pat. Nos. 4,986,964; 5,293,772 and 5,205,159 directly measures the force developed by platelets during clot retraction. Carr's apparatus consists of a cup in which the fluid sample (before clotting) is placed. The opening of the cup is covered by an upper plate, which is coupled to a steel arm attached to a force transducer. As the clot retracts, the force generated is transmitted to the force transducer, where it is measured (1;4;13;14). Although this is a very reliable method, the cost per measurement is high, because this method allows only the measurement of one sample at a time. Also, this equipment has the added complication of the high precision required for its alignment and setup.
Therefore, it would be advantageous to have a low-cost, reliable method for the quantitation and monitoring of the force developed during clot retraction. This would provide a simple way to assess several variables of clinical relevance that converge into one single measurable variable, i.e. using the above mentioned funnel detection philosophy. Moreover, what is needed is an easy-to-use and economical device to accurately measure the force developed during clot retraction. This device should be self-contained in order to minimize exposure to biohazardous materials. Such a device would have a broad spectrum of clinical applications, including, for example, patient evaluation and population screening for pathological conditions.